Haynes:WestBlot

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Western Blot

by Karmella Haynes, 2015
Antibody staining - "floating" technique adapted from R. Tirabassi, Bitesizebio blog

Principle: Proteins that have been separated by size on a polyacrylamide gel (by PAGE)are transferred onto a solid paper-like membrane, such as nitrocellulose. One specific protein is visualized by incuabating the membrane with labeled antibodies, then imaging the membrane. A visible band will appear that corresponds to the size of the protein.

MATERIALS

• PAGE gel containing electrophoresed protein samples
• Tris-glycine concentrate [250 mM Tris; 1.92 M glycine; pH approx. 8.3] (see recipe)
• Methanol
• Blotting paper/ "filter" paper (e.g. Bio-Rad Nitrocellulose & filter sets 1620212)
• Blotting membrane - nitrocellulose or PVDF (e.g., Bio-Rad Nitrocellulose & filter sets 1620212)
• 10x PBS
• Tween-20 (e.g., G-Biosciences 786-517)
• 0.01% Ponceau-S Stain
• Nonfat dry milk
• Primary antibody
• Secondary antibody
• HRP detection reagents (IF using an HRP secondary antibody)

EQUIPMENT

• Electroblotting chamber (e.g. Invitorgen XCell SureLock)
• 4 blotting sponge pads (e.g. Invitrogen EI9052) - usually included with electroblotting unit

PROCEDURE

Prepare 1x Transfer Buffer [12.5 mM Tris; 96 mM glycine; 10% methanol]

1. Add ~200 mL dH₂O, 25 mL Tris-glycine concentrate, and 50 mL methanol to a 500 mL bottle. Swirl to mix.
2. Fill up to 500 mL with dH₂O (+ ~225 mL dH₂O). Note: deionized water from the tap is sufficient.

Prepare a gel and filter stack

Instructions for a wet chamber - handle materials with gloved hands. Use forceps only for the blotting membrane.

1. Remove the gel from the mold. Ue a scalpel to trim the lanes off the top and the rough edge off the bottom. Soak in a small dish of 1x Transfer buffer “back” side up. Note: the proteins will migrate "backward" onto the nitrocellulose filter.
2. In a small flat tupperware-like or glass container of 1x Transfer Buffer, wet two blotting sponge pads. Stack one on top of the other.
3. Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of the two wet, stacked sponge pads.
4. Carefully place the gel carefully onto the wet blotting paper. Make sure the gel is "back" side up.
5. IMPORTANT: Use a smooth "roller" (e.g. a clean, short piece of a plastic graduated or aspiration pipette) to roll away any bubbles that may be trapped between the blotting paper and gel. Note, bubbles will block protein transfer.
6. Take the blotting membrane and cut off a small piece of one of the corners with a pair of scissors.
7. Wet the blotting membrane in the 1x Transfer buffer. Orient the cut corner so that it is on the bottom right. Carefully and gradually (starting at one edge) lay it on top of the gel (to avoid bubbles).
8. Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of the blotting membrane.
9. IMPORTANT: Use a smooth "roller" (e.g. a clean, short piece of a plastic graduated or aspiration pipette) to roll away any bubbles that may be trapped between the gel and blotting membrane. Note, bubbles will block protein transfer.
10. Wet the remaining two blotting sponge pads. Stack these on top of the blotting paper.

REMEMBER: From bottom to top, it's Sponge-Sponge-Paper-Gel-Membrane-Paper-Sponge-Sponge

Prepare the electrophoresis set-up

1. Place the gel and filter stack into the elctroblotting module/holder so that the membrane is closest to the positive electrode side and the gel is closets to the negative electrode side.
2. Insert this assembly into the electrophoresis tank with the positive and negative electrode sides in the proper orientation. Note: If using the Invitrogen XCell SureLock system, use the clamp to secure the blotting module.
3. Fill the tank with the appropriate solution(s)
   1. XCell SureLock: fill the inner chamber with 1x Transfer Buffer. Fill the outer chamber with deionized water, about halfway.
   2. Other systems: typically 1x Transfer Buffer for the whole tank, plus a frozen ice pack to keep the system cooled down.
4. Connect the electrodes to the chamber and a power supply.
5. Run the transfer...
   1. Invitrogen XCell SureLock - 30 volts for 1 hour.

Prepare 1x PBS-tween [? mM Na2HPO4; ? mM NaH2PO4; ? nmM NaCl; 0.1% Tween-20; pH ~7.2]

1. Add ~200 mL dH₂O, 50 mL 10x PBS, and 0.5 mL Tween-20 to a 500 mL bottle. Swirl to mix.
2. Fill up to 500 mL with dH₂O (+ ~249.5 mL dH₂O). Note: deionized water from the tap is sufficient.

OPTIONAL: Visualizes transferred protein with Ponceau-S

1. Rinse the membrane protein-side-up in dH₂O.
2. Soak in 1X Ponceau (just enough to cover the membrane) until bands appear.
3. Wash off excess with dH₂O. Record an image.
4. Mark the protein ladder in pencil.
5. Wash off Ponceau staining with 1x PBS-tween.

Antibody staining (primary) - "floating" technique (overnight step)

1. Prepare blocking buffer [5% nonfat dry milk in 1x PBS-tween]: In a small bottle (~200 mL) add 5.0 g nonfat dry milk to 100 mL 1x PBS-tween. Note: Only make a very small amount. Leftover solution is only good for 48 hours, stored at 4°C.
2. Incubate membrane in Blocking solution at romm temp. for > 1 hr with gentle rocking. Note: blocking can be done overnight at 4°C.
3. Cut a piece of parafilm that is slightly larger than the membrane.
4. Obtain a large (25 cm) petri dish or other sealable, clean, flat-bottomed container.
5. Pipette about 2 mL blocking buffer into the dish and spread it around with a Kimwipe.
6. Lay the parafilm onto the wetted area without creating bubbles between the parafilm and the container. This sticks the parafilm to the bottom of the container.
7. Wipe off excess blocking buffer from around the parafilm.
8. Dilute primary antibody in blocking buffer (see your ab specs for recommended dilution). Use 1mL for a full membrane (9×5.5 cm), and 0.5 mL for half a membrane.
9. Pipette diluted antibody onto the parafilm near one end.
10. Using forceps, pick up membrane that has been incubated in blocking buffer and lay protein side down onto the diluted antibody. I lay one edge of the membrane against the liquid and allow the membrane to wick across. Keep the membrane on the parfilm and be careful to prevent any bubbles from forming between the liquid and the membrane.
11. Seal container with lid and parafilm if necessary. This must be an air-tight seal or the diluted antibody will evaporate.
12. Incubate with primary antibody using conditions optimized for the antibody (typically 4°C overnight).

Antibody staining (secondary) - "floating" technique

1. Transfer the membrane into a new container for washes.
2. Wash membrane in 1x PBS-tween at room temp. for 15 min., 4 times.
3. Repeat the procedure as described in Antibody staining (primary) - "floating" technique, except...
   1. Use the secondary antibody. Dilution is typically 1:10,000 for an HRP-conjugated antibody.
4. Incubate with secondary antibody using conditions optimized for the antibody. Incubation is typically at room temperature for no more than 1 hour. Do not over-incubate!

Antibody detection

HRP - SuperSignal West Femto Substrate

1. Transfer the membrane into a new container for washes.
2. Wash membrane in 1x PBS-tween at room temp. for 15 min., 4 times.
3. tbc

HELPFUL RESOURCES

• Electroblotting wet transfer: Video - How to perform a traditional wet protein transfer using the XCell SureLock® Blot Module